Entomopathogenic nematodes are important biological control agents for a variety of economically important pests in agricultural and urban environments (Grewal and Georgis, Entomophathogenic nematodes, “Methods in Biotechnology”, Volume 5, Biopesticides: Use and Delivery, F. R. Hall and J. J. Menn, Eds., 271-299, 1998, Totowa, N.J., Humana Press, Inc.; Kaya and Gaugler, Entomopathogenic Nematodes, Annu Rev. Entomol., Volume 38, 181-206, 1993). Specific entomopathogenic nematodes, Steinernema and Heterorhabditis, are important biological control agents for a variety of economically important pests. The nematodes can be mass-produced using in vivo or in vitro methods (Shapiro-Ilan, D. I., Gaugler, R., 2002. Production technology for entomopathogenic nematodes and their bacterial symbionts. J. Ind. Microbiol. & Biotech. 28, 137-146; Ehlers, R-U., Shapiro-Ilan, D. I., 2005. Mass production. In: Grewal, P. S., Ehlers, R-U, Shapiro-Ilan, D. I. (Eds.), Nematodes as Biocontrol Agents. CABI Publishing, Wallingford, UK, pp. 65-78). Additionally, U.S. Pat. No. 6,474,259 discloses in vivo, apparatus and methods for producing insecticidal nematodes resulting in the nematodes harvested into an aqueous solution.
Current commercial entomopathogenic nematodes are generally applied as infective juveniles (Us) in aqueous suspensions using various irrigation systems, sprayers, or injection techniques (Grewal, P. S., 2002. Formulation and application technology. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Wallingford, UK, pp. 265-288; Creighton, C. S., Fassuliotis, G., 1985. Heterorhabditis sp. (Nematoda: Heterorhabditidae): A nematode parasite isolated from the banded cucumber beetle Diabrotica balteata. J. Nematol. 17, 150-153).
Entomopathogenic nematodes may also be applied in infected insect arthropods (Creighton, C. S., Fassuliotis, G., 1985. Heterorhabditis sp. (Nematoda: Heterorhabditidae): A nematode parasite isolated from the banded cucumber beetle Diabrotica balteata. J. Nematol. 17, 150-153; Jansson, R. K., Lecrone, S. H, Gaugler, R., 1993. Field efficacy and persistence of entomopathogenic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae) for control of sweetpotato weevil (Coleoptera: Apionidae) in southern Florida. J. Econ. Entomol. 86, 1055-1063; Shapiro-Ilan, D. I., Lewis, E. E., Tedders, W. L., Son, Y., 2003. Superior efficacy observed in entomopathogenic nematodes applied in infected-host arthropods compared with application in aqueous suspension. J. Invertebr. Pathol. 83, 270-272; Bruck, D. J., Shapiro-Ilan, D. I., Lewis, E. E., 2005. Evaluation of application technologies of entomopathogenic nematodes for control of the black vine weevil, Otiorhynchus sulcatus. J. Econ. Entomol. 98, 1884-1889; Del Valle, E. E., Dolinksi, C., Barreto, E. L. S., Souza, R. M., Samuels, R. I., 2008. Efficacy of Heterorhabditis baujardi LP77 (Nematoda: Rhabditida) applied in Galleria mellonella (Lepidoptera: Pyralidae) insect arthropods to Conotrachelus psidii (Coleoptera: Curculionidae) larvae. Biocontrol Sci. Technol. 18, 33-41). In this approach, nematode-infected arthropods are disseminated and pest suppression is subsequently achieved by the progeny infective juveniles that exit the arthropods. Laboratory studies indicate that nematode application in infected hosts may be superior to application in aqueous suspension (Shapiro, D. I., Glazer, I., 1996. Comparison of entomopathogenic nematode dispersal from infected hosts versus aqueous suspension. Environ. Entomol. 25, 1455-1461; Shapiro, D. I., Lewis, E. E., 1999. Comparison of entomopathogenic nematode infectivity from infected hosts versus aqueous suspension. Environ. Entomol. 28, 907-911; Perez, E. E., Lewis, E. E., Shapiro-Ilan, D. I., 2003. Impact of host arthropod on survival and infectivity of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) under desiccating conditions. J. Invertebr. Pathol. 82, 111-118). Additionally, pest control trials have indicated that arthropod application can be superior in efficacy to aqueous application (Shapiro-Ilan, D. I., Lewis, E. E., Tedders, W. L., Son, Y., 2003. Additionally, superior efficacy was observed in entomopathogenic nematodes applied in infected-host arthropods compared with application in aqueous suspension. J. Invertebr. Pathol. 83, 270-272).
While arthropod applications have been shown to be advantageous over aqueous applications, a potential problem with the arthropod applications is that infected hosts can rupture or stick together during transport or distribution (Shapiro-Ilan, D. I., Lewis, E. E. Behle, R. W., McGuire, M. R., 2001. Formulation of entomopathogenic nematode-infected-arthropods. J. Invertebr. Pathol. 78, 17-23). As disclosed in U.S. Pat. No. 6,524,601, the problem of rupturing arthropods can be ameliorated in soft bodied hosts such as the greater wax moth, Galleria mellonella (L.) (Lepidoptera: Pyralidae), by coating the arthropods with a powder such as clay. Similarly, arthropod coatings were developed for application of Heterorhabditis baujardi Phan et al. (Del Valle et al., 2009). Another approach is to use hard-bodied insects, such as the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae), for which the harder cuticle can naturally resist rupture and prevent arthropods from sticking together. Yet, use of hard-bodied infected hosts may still result in some rupturing, and furthermore use of arthropods in certain markets, such as home gardens, may be limited due to an aversion of touching insects (Kellert, S. R., 1993. Values and perceptions of invertebrates. Conservation Biol. 7, 845-855). Thus, there is a need to develop a formulation that protects arthropods and allows ease of handing may be beneficial for hard and soft bodied hosts. Additionally, a formulation that is amenable to mass production and standardization will facilitate successful commercialization of utilizing nematodes as a biological control method.
Additionally there is a cost-benefit in applying nematode in infected arthropods as compared to aqueous nematode applications inasmuch as the former requires less steps in the process and is thus less costly (Shapiro-Ilan and Gauger, 2002, supra). Furthermore, it has been reported that entomopathogenic nematodes can survive dry conditions for extended periods if they remain inside a host arthropod. Commercialization of nematode-infected arthropods has been prevented due to problems in storage and application (Koppenhofer, A., “Nematodes”, Field Manual of Techniques in Invertebrate Pathology, Chapter 4-5 pg. 283-301, 2000). Nematode-infected hosts stick together or rupture during transport and/or application, which results in reduced efficacy. As such there is a need for confer additional protection to infected hosts arthropods to minimize disruption during transport and application of the biological control.